Get access

Application of high-resolution melting for genotyping bovine mitochondrial DNA

Authors

  • Zai-Wei Zhou,

    1. Shanghai Institute of Medical Genetics, Shanghai Children's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
    Search for more papers by this author
  • Jing-Bin Yan,

    1. Shanghai Institute of Medical Genetics, Shanghai Children's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
    2. Key Laboratory of Embryo Molecular Biology, Ministry of Health, China
    3. Shanghai Laboratory of Reproduction and Embryo Engineering, Shanghai, China
    Search for more papers by this author
  • Hua Li,

    1. Shanghai Institute of Medical Genetics, Shanghai Children's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
    2. Key Laboratory of Embryo Molecular Biology, Ministry of Health, China
    3. Shanghai Laboratory of Reproduction and Embryo Engineering, Shanghai, China
    Search for more papers by this author
  • Zhao-Rui Ren

    Corresponding author
    1. Shanghai Institute of Medical Genetics, Shanghai Children's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
    2. Key Laboratory of Embryo Molecular Biology, Ministry of Health, China
    3. Shanghai Laboratory of Reproduction and Embryo Engineering, Shanghai, China
    • Key Laboratory of Embryo Molecular Biology, Shanghai, China
    Search for more papers by this author

Abstract

Recent studies have demonstrated that mitochondrial DNA (mtDNA) haplotype has a significant impact on the efficiency of bovine somatic cell nuclear transfer. Conventional methods for detecting mtDNA variations and haplotypes, such as restriction fragment length polymorphism (RFLP), temporal temperature gradient gel electrophoresis, dHPLC and sequencing, are labor intensive or expensive and have low sensitivity. High-resolution melting (HRM) analysis is a new technique for mutation detection and has the advantages of speed, cost, and accuracy. Here, we describe the genotyping of bovine mtDNA using HRM analysis. DNA samples containing mtDNA were extracted from 75 Holstein cows and subjected to rapid-cycle (<20 min) PCR of small amplicons (<120 bp) using specific primer sets. Capillaries containing the PCR products were then subjected to HRM analysis; data were acquired in 2 min and analyzed using the instrument's software. Five common bovine mtDNA single nucleotide polymorphisms were identified: 9602 G>A, 169 A>G, 166A>G with 173A>G, and 363C>G. These results agree with both sequencing and RFLP analysis. In addition, a very small amount of heteroplasmic variants (<5%) was sufficiently to be distinguished by HRM analysis that would be very useful to differentiate heteroplasmy vs. homoplasmy. HRM analysis thus provides a new approach to genotyping bovine mtDNA sequence variations and has many advantages over other methods, including speed of analysis, cost, and accuracy. We believe this will be a valuable technique for determining the efficiency of nuclear transfer in cloned embryos and for studying maternal effects on nuclear-cytoplasm interactions. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011

Get access to the full text of this article

Ancillary